Radiolabelled leucocytes in human pulmonary disease

Abstract

Introduction

Radionuclides for leucocyte kinetic studies have progressed from non-gamma emitting cell-labelling radionuclides through gamma emitting nuclides that allow imaging of leucocyte kinetics, to the next goal of positron emission tomography (PET).

Sources of data

Mostly the authors’ own studies, following on from studies of the early pioneers.

Areas of controversy

From early imaging studies, it appeared that the majority of the marginated granulocyte pool was located in the lungs. However, later work disputed this by demonstrating the exquisite sensitivity of granulocytes to ex vivo isolation and labelling, and that excessive lung activity is artefactual.

Areas of agreement

Following refinement of labelling techniques, it was shown that the majority of marginated granulocytes are located in the spleen and bone marrow. The majority of leucocytes have a pulmonary vascular transit time only a few seconds longer than erythrocytes. The minority showing slow transit, ~5% in healthy persons, is increased in systemic inflammatory disorders that cause neutrophil priming and loss of deformability. Using a range of imaging techniques, including gamma camera imaging, whole-body counting and single photon-emission computerized tomography, labelled granulocytes were subsequently used to image pulmonary trafficking in lobar pneumonia, bronchiectasis, chronic obstructive pulmonary disease and adult respiratory distress syndrome.

Growing points

More recently, eosinophils have been separated in pure form using magnetic bead technology for the study of eosinophil trafficking in asthma.

Areas timely for developing research

These include advancement of eosinophil imaging, development of monocyte labelling, development of cell labelling with PET tracers and the tracking of lymphocytes.

Introduction

The early blood cell labels, P-32-labelled di-isopropylfluorophosphate (DFP-32) and H-3, are non-gamma emitters and cannot be imaged, so early work with radiolabelled leucocytes was limited to kinetic studies based on counting radioactivity in sampled blood. These studies, nevertheless, were seminal and established the residence time of granulocytes in blood and their distribution between circulating and marginating pools. Cr-51 labelling was a step towards imaging but, because this radionuclide is an inefficient emitter of gamma photons, moreover at too high an energy for imaging, only surface counting over organs with scintillation probes could be used to determine the in vivo distribution of labelled cells. In the mid-seventies, Matthew Thakur, a radiochemist at Hammersmith Hospital, and John McAfee, a visiting nuclear medicine physician from USA, achieved their aim, within McAfee’s 6 month sabbatical, of labelling blood cells, including leucocytes, with the efficient gamma emitter, In-111, thereby allowing imaging of infection and inflammation and determination of granulocyte kinetics. It was several years later before Tc-99m labelling was developed to complement In-111 labelling. Lymphocytes, however, remained problematic because of their exquisite radiosensitivity.

Techniques

Labelling leucocyte subsets involves isolation of the cell of interest followed by exposure to a radionuclide that penetrates and labels the cell. Subset nomenclature may be confusing. Labelled ‘leucocytes’ comprise a mixture of all blood cells, enriched with leucocytes and generally used clinically. Labelled granulocytes comprise neutrophils and eosinophils. As the neutrophils usually greatly outnumber eosinophils, labelled ‘granulocytes’ are essentially the same as labelled ‘neutrophils’. More recently, through magnetic bead technology, eosinophils have been isolated in pure form. Neutrophils can also be further purified by removal of eosinophils, but this is generally not necessary, even for research studies. Basophils are too scarce to be a practical consideration.

Cell separation

In early studies using DFP-32, whole blood was labelled, while H-3-thymidine was used to label cells as they developed in vivo. Cell-associated activity was then determined in cells isolated from post-injection blood samples. In later studies based on imaging, it was necessary to first isolate the blood cell of interest. Leucocyte isolation starts with erythrocyte sedimentation from anti-coagulated peripheral venous whole blood using sedimenting agents such as hydroxyethyl starch. Sedimentation leaves a supernatant containing platelets, non-sedimented erythrocytes and all leucocyte subtypes. For routine clinical use, this supernatant is centrifuged slowly, so that the majority of platelets remain in suspension, to give a pellet of cells, which is then re-suspended in a small volume of appropriate medium for exposure to the radionuclide. The majority of the radioactivity ends up in leucocytes, although this varies according to the peripheral blood count, and the preparation is referred to as ‘mixed leucocytes’.

The mixture of leucocyte subtypes can be further purified using density gradient centrifugation, which separates mononuclears (largely lymphocytes and monocytes) from granulocytes. Percoll diluted with autologous plasma is the preferred gradient solution, as will be evident below.^1–3 Granulocytes can be further separated into neutrophils and eosinophils, and monocytes separated from lymphocytes, using magnetic bead technology, which is based on monoclonal antibodies attached to magnetic beads.^3–5 The leucocyte of interest is isolated by negative or positive selection, using an appropriate antibody. Negative selection is preferable because of the greater likelihood of cell activation resulting from antibody and bead attachment.

Cell radiolabelling

In contrast to Cr-51, which is used to label blood cells, including leucocytes, as sodium chromate, In-111 and Tc-99m in ionic form cannot permeate cell membranes and must first be complexed to a lipophilic chelating agent for cell labelling. Hexamethylpropyleneamine oxime (HMPAO) is generally used for Tc-99m labeling, and oxine (now commercially unavailable) or tropolone for In-111. In-111 is retained in leucocytes as a result of strong binding to intracellular proteins, while Tc-99m is retained as a result of intracellular conversion, involving glutathione, to a non-lipophilic complex. In-111-chelates have more or less equal affinity for granulocyte subtypes but Tc-99m–HMPAO displays strong selectivity for eosinophils (20-fold more activity binds eosinophils compared with equal numbers of neutrophils).⁶

Cr-51-labelling is now essentially obsolete. In a clinical setting, the choice between In-111 and Tc-99m is largely driven by the indication, for example In-111 for orthopaedic infections, which are largely neutrophilic, and Tc-99m for inflammatory bowel disease (IBD), which is significantly eosinophilic.⁷ The time-course of planned studies is also relevant, especially for experimental studies. Thus, Tc-99m has a shorter physical half-life (6 h) but is photon emission-rich and therefore well suited to short-term kinetic studies, while In-111 has a longer half-life (68 h) and labels cells with high stability, and is therefore better suited to longer-term studies, such as measurement of intravascular neutrophil life-span.

It is noteworthy that in the era of positron emission tomography/computed tomography (PET/CT), no positron-emitting radionuclides have yet impacted on cell labeling because no PET complex has been found that stably labels blood cells. Activated inflammatory cells accumulate the glucose analogue F-18-fluorodeoxyglucose (FDG) as a result of their increased metabolic activity, and FDG on its own is used routinely to image inflammation, without there necessarily being knowledge of the cells responsible for tracer accumulation. Attempts have therefore been made to label leucocytes ex vivo with FDG, but the cell activation necessary to encourage uptake of tracer inevitably results in subsequent un-physiological in vivo kinetics. Moreover, the labelling is insufficiently stable and the physical half-life of F-18 of only 109 min is too short-lived for longer-term studies.

Assessing cellular integrity following labelling

Isolating cells in vitro and subjecting them to labeling agents inevitably leads to disturbance in their cellular integrity as a result of mechanical, chemical and radiation toxicity. Lymphocytes in particular are highly radiosensitive and do not survive in a mixed leucocyte preparation. Cellular integrity following labelling can be assessed by various laboratory methods, the simplest and most described of which is the ability of the cell to exclude vital dyes, such as trypan blue. This however only proves the cell is alive. The most sensitive benchmark is in vivo recovery, which is cell-bound activity as a percentage of administered activity in a peripheral venous blood sample taken 30–45 min after injection. For neutrophils, recovery is optimally about 40–50%, although, as it reflects the size of the marginating neutrophil pool, it is higher in splenectomised individuals.

Scintigraphy

The in vivo distribution of radiolabelled cells can be detected and quantified using various detectors, including scintillation probes, gamma cameras and whole-body counters.

Scintillation probe

Collimated scintillation probes may be placed on the surface of the skin to detect local radioactivity (surface counting). Such probes were used to semi-quantify Cr-51-labelled leucocyte deposition in spleen, liver and pelvic bone marrow, and continue to be used in whole-body counters.

Gamma camera

Planar static and dynamic imaging

A gamma camera gives a two-dimensional (planar) image of the location of radioactivity. In dynamic imaging, multiple image ‘frames’ of between 1 s (at the start) and 60 s (towards the end) each, are continuously acquired over time following injection, typically 30–40 min. The time-course of radioactivity enables measurement of the transit times of neutrophils across the pulmonary vasculature and other vascular beds, such as liver and spleen.

Single photon-emission computed tomography

In SPECT, multiple images are acquired as the gamma camera undergoes 360° rotation around the subject, enabling generation of a 3-dimensional image. SPECT of the lungs is important in research studies on pulmonary granulocyte kinetics because it separates physiological activity in bone marrow of ribs, sternum and vertebrae from pulmonary activity, a distinction that is not possible with planar imaging. Co-registered low-dose CT allows precise anatomical localization of radioactivity (SPECT/CT).

Whole-body counter

Whole-body counting provides no image resolution but instead enables profiling of regional distribution of radiolabelled cells. Highly sensitive scintillation probes move from the subject’s head to toe, generating a longitudinal radioactivity profile (Fig. 1). The counter is heavily shielded from background radiation and provides high photon detection sensitivity, thereby allowing the use of very low levels of administered radioactivity.

Fig. 1.

Measurement of whole-body neutrophil distribution in a dedicated whole-body counter. (A) Photograph of a lead-lined whole-body counter. The lead shielding excludes background radiation and allows detection of ultra-low levels of administered activity. The anterior (ANT) and posterior (POST) gamma detectors are labelled. (B) Schematic of a subject lying supine within a whole-body counter (upper panel). The subject is positioned between anterior and posterior scintillation detectors (γ) that move simultaneously from head to toe. The lower panel illustrates a representative whole-body profile 24 h following injection of <0.5 MBq In-111-labelled neutrophils. Panel B was published by Lok et al.⁴⁸

Sample counting

Concentration of radioactivity in fluid samples can be measured using a well-counter. Blood sample counting is used to determine recovery and subsequent intravascular lifespan of labelled cells. Measurement of radioactivity in sputum and faeces has also proved useful for quantification of cell migration at appropriate sites.

Image analysis

Planar static imaging

To semi-quantify local radioactivity, regions of interest (ROI) are placed over organs of interest to obtain counts/pixel/administered activity. Correction for photon attenuation in tissue can be made if the depth of the tissue is known.

Planar dynamic imaging

Several methods are available for analyzing dynamic imaging data. For example, tissue intravascular leucocyte transit time can be calculated by fitting a gamma function to the individual frame values or by using deconvolution analysis, which compares tissue activity with activity in blood. Gjedde–Patlak–Rutland graphical analysis is also based on activities in tissue and blood but develops the mathematics further to measure blood clearance of activity into tissue.⁸ In graphical analysis, the ratio of tissue-to-blood activity is plotted against ‘normalized time’ (integral [running sum] of blood counts divided by instantaneous blood counts). The gradient of the plot (Ki) represents tissue blood clearance while the intercept represents the distribution volume (V0) of the tracer within the tissue. For neutrophils in the lung, V0 represents the lung marginated neutrophil pool. Without knowledge of absolute tissue and blood tracer concentrations, Ki and V0 are not quantifiable, but because the proportionality constants respectively relating their absolute concentrations to count rates are identical, Ki divided by V0 measures clearance normalized to distribution volume. This is attractive for the lungs because variability in air volume effectively renders lung tracer concentration somewhat meaningless.

Whole-body counting

The whole-body profile in a patient injected with In-111-labelled neutrophils displays several peaks, corresponding to chest, hepatosplenic region and pelvic bone marrow (Fig. 1). The profile changes as a result of the label undergoing re-distribution over time. Because of the great sensitivity of whole-body counting, profiles can be recorded for up 10 days post-injection, even following administration of tiny doses of In-111. Few departments have whole-body counters, but an alternative is a gamma camera without its collimator. This, however, is less sensitive and requires larger administered activities.

SPECT

Using CT for attenuation, SPECT has the potential, like PET, to quantify tissue In-111 and Tc-99m activities as absolute concentrations (MBq/ml). We are not aware, however, of such analysis being applied to labelled leucocytes. Instead, Gjedde–Patlak–Rutland graphical analysis has been applied to sequential SPECT to measure blood granulocyte clearance into the lungs.

Pathophysiology of neutrophil kinetics in lungs

Early evidence that neutrophils are delayed physiologically in the lungs

The concept of circulating, marginating and total blood granulocyte pools arose from the pioneering work of Wintrobe’s group in the 1960s, who showed that only about 50% of labelled granulocytes (i.e. the recovery) could be accounted for in peripheral blood following intravenous injection of a crude mixture of blood cells labelled with DFP-32.⁹ This led to the view that 50% of granulocytes were ‘marginated’ as a result of adherence to the endothelium of vascular beds throughout the body. The concept of margination and demargination was re-enforced by finding that the recovery could be increased by exercise or the administration of adrenaline.⁹ Dancey et al., using H-3-thymidine as an in vivo label, confirmed the presence of a marginated pool of about 50%.¹⁰ This early work also established that granulocytes leave the circulation exponentially with a half-time of about 7 h (corresponding to a mean intravascular residence time of 10 h).^10,11 McMillan and Scott recorded almost identical half-times for neutrophils respectively labelled with Cr-51 (10.5 h) and DFP-32 (10.3 h) but obtained a lower recovery with Cr-51 (20% versus 56%).¹² Like Wintrobe et al., they obtained evidence of demargination following adrenaline administration, and, moreover, using surface counting demonstrated simultaneous release of label from the spleen. A similar blood survival half-time was later confirmed with In-111-labelled neutrophils.¹³ More recent work with deuterium-labelled granulocytes, however, suggested that intravascular granulocyte lifespan may be much longer (~5 days),¹⁴ but this finding has been criticized.¹⁵ Eosinophils have a longer intravascular lifespan than neutrophils of about 25 h and a lower recovery of about 15%, implying a larger marginating pool.³

With the development of cell labelling with In-111, the sites of margination could be imaged. As a result, the concept of widespread endothelial adherence was undermined and instead it was thought from experiments, mainly in dogs and rabbits, that the pulmonary vascular bed was the principal site of margination, with claims that up to 90% of the total blood granulocyte pool resides in the lungs.¹⁶

Experience arising from clinical imaging with labelled leucocytes

Labelling of purified neutrophils is considered too labour-intensive for routine clinical work, for which mixed leucocytes are generally used. Early observations of prominent lung activity immediately following injection were consistent with the view that the lungs are an important site for neutrophil margination. However, as a result of further studies in which the labelling conditions were critically examined (e.g. labelling in plasma-enriched media versus saline), it became clear that prominent early lung activity was not physiological but instead the result of some form of injury to or activation of the labelled cells, and therefore came to be regarded as a marker of quality control of the labelling (Fig. 2).¹⁷ This is not to say that the lung is not an important site of physiological margination, but rather that its role had been earlier over-emphasized. Other sites emerged where granulocytes were shown to undergo more prolonged temporary residence, specifically bone marrow and spleen, and, to a lesser extent, the liver. The physiological mechanisms underpinning margination are largely unknown, although in the lungs, capillaries have a critical cross-sectional diameter that requires neutrophils to deform in order to negotiate. So the physiological distribution of activity following injection of In-111-labelled leucocytes or purified granulocytes is primarily spleen and bone marrow with less prominent activity in the liver and lungs. At 24 h, when almost all the labelled cells have left the circulation, the sites of activity are liver, spleen and marrow, which are the sites of physiological neutrophil disposal (Fig. 3).

Fig. 2.

Pulmonary transit of labelled granulocytes – influence of labelling technique. Planar gamma camera images acquired soon after injections of granulocytes isolated from preparations of mixed leucocytes using Percoll-plasma (upper panels) or Percoll-saline (lower panels) and then labelled with In-111. Upper panels show immediate and 5 min post-injection images. Note rapid transit of cells through the pulmonary vasculature (LL: left lung; RL: right lung), prominent splenic (S) activity and modest hepatic (L) activity. Cardiac blood pool activity is clearly visible at 5 min. Lower panels, in contrast, show lung activity persisting beyond 40 min and minimal splenic activity (S). Hepatic activity (L) is also minimal but increases markedly as activity is released from lungs (not shown). Note that lower panel images are from posterior projection so spleen is on the left and liver on the right.

Fig. 3.

Neutrophil priming in systemic inflammation. In-111-leucocyte scan in a patient with upper abdominal sepsis (dark blue arrow at 24 h post-injection, also faintly visible at 3 h) complicating acute pancreatitis. Note radioactive pus in drainage bags (red arrows). There is prominent lung activity at 3 h post-injection that clears by 24 h, leaving only physiological chest activity within bone marrow of sternum, ribs and spine. Anterior (ANT) and posterior (POST) images are shown. Distributions of activity in liver, spleen and bone marrow are otherwise normal.

Circumstances in which neutrophil transit time in the lungs is patho-physiologically prolonged include neutrophil priming, which results in neutrophil shape change and reduced deformability, and local lung conditions such as pleural effusion that compress lung capillaries. Neutrophil priming is seen in several severe inflammatory conditions,¹⁸ including systemic vasculitis, graft-versus-host disease, severe IBD, severe alcoholic hepatitis and pancreatitis, and gives rise to a characteristic image of early prominent lung activity that ‘clears’ by 24 h (Fig. 3). Expressing it as a proportion of total blood granulocyte pool, Ussov et al. measured the size of the pulmonary granulocyte pool from count rates recorded over the lung 15–30 min post-injection of labelled granulocytes and found it to be increased in systemic inflammatory conditions (e.g. 0.34 in systemic vasculitis compared with 0.08 in controls).¹⁹ Priming does not result in reduced splenic and marrow activities, in contrast to neutrophils that are artefactually stimulated as a result of suboptimal labelling technique, which give low levels of marrow and spleen uptake and prominent hepatic uptake.

Intravascular pulmonary transit time of granulocytes

Using fluorescence video-microscopy in dogs, Lien et al.²⁰ directly observed fluorescein isothiocyanate-labeled neutrophils in transit through the pulmonary vasculature. Most neutrophils passed rapidly in a few seconds but a variable minority was discretely immobilized in pulmonary capillaries for periods of up to 20 min. In keeping with the concept of a bimodal distribution of transit times, Ussov et al.²¹ measured lung granulocyte transit time using deconvolution analysis of dynamic data in healthy subjects and patients with inflammatory disease and identified a fast fraction with a transit time of 20–25 s, irrespective of pathology, compared with 4.3 s for erythrocytes. The fraction displaying slower transit, recorded as 120–138 s and again independent of pathology, was 11% in controls compared with 32% in systemic vasculitis, and correlated with the fraction of peripheral venous neutrophils that were primed. Measuring the areas under the arterial outflow time-concentration curves following co-injection of In-111-labelled neutrophils and Tc-99m-labelled erythrocytes into the central venous circulation in dogs, Hogg et al. recorded a first pass retention of 85% of neutrophils, a very high value compared with Ussov et al., that was likely the result of labeling neutrophils in saline.²² Pursuing a similar dual labelling technique in healthy humans but instead labelling neutrophils in plasma, Summers et al.¹ recorded a retention of only about 5% and showed that the ~95% displaying fast transit had a mean trans-pulmonary transit time only 2.3 s slower than that of erythrocytes. From dynamic planar scintigraphy in healthy subjects, they recorded a mean pulmonary neutrophil transit time of ~14 s. In contrast, when neutrophils had been primed ex vivo with GM-CSF prior to injection, 96% were retained in the lungs for at least 2 min. Retention was also increased following ex vivo priming with platelet activating factor (PAF). PAF, however, primes neutrophils reversibly and, accordingly, lung neutrophil retention fraction returned to control values when re-infusion of the cells was delayed by 30 min.¹

Inhalation of PAF as an aerosol has also been shown to cause neutrophil accumulation in the lungs,²³ probably as a result of neutrophil priming in situ, as PAF, being lipophilic, would rapidly enter pulmonary blood following inhalation and prime neutrophils as they passed. MacNee at al found that inhalation of cigarette smoke during dynamic imaging following injection of In-111-labelled neutrophils produced a more slowly declining lung signal. Although interpreted as smoke-induced prolongation of pulmonary neutrophil transit time,²⁴ a reduction in splenic pooling caused by the smoke, which caused nausea, could not be excluded as an alternative explanation.

There is currently no evidence to suggest that increased pulmonary neutrophil transit time is the direct result of activation of pulmonary vascular endothelium. Instead, overall mean pulmonary neutrophil transit time, and therefore the total lung content of transiting neutrophils, appears to depend on the proportion of neutrophils displaying slow transit and can be measured from static imaging after the labelled neutrophils have mixed in the lung marginated pool.¹⁹ By comparing count rates over the lungs with count rates in peripheral blood, Peters et al. expressed neutrophil transit time in relation to erythrocyte transit time from a preceding injection of erythrocytes labelled with the same radionuclide.²⁵ Using this approach, overall mean In-111-neutrophil transit time was ~2.5-fold that of In-111-erythrocytes. An erythrocyte transit time of 6 s (which assumes a cardiac output of 5 l/min and lung blood volume of 500 ml) would indicate a mean overall granulocyte transit time of 15 s.

Radiolabelled leucocytes in lung diseases

When first introduced to clinical practice in the early eighties, labelled leucocytes made a major impact in the diagnosis of a range of infectious and inflammatory conditions, especially soft tissue abscesses. Within a few years, the commonest clinical indication for leucocyte scintigraphy was IBD. As other diagnostic techniques have developed in the management of abscesses and IBD, the commonest clinical indication of leucocyte scintigraphy nowadays is orthopaedic infections, especially of prosthetic joints. In contrast, radiolabelled leucocytes have never been widely used in the routine management of lung diseases, despite the important roles of neutrophils in their pathogenesis. This has not, however, prevented the use of labelled neutrophils to investigate pathophysiology in several important inflammatory lung diseases, as illustrated in the following sections.

Acute respiratory distress syndrome

Acute respiratory distress syndrome (ARDS) is associated with refractory hypoxaemia and neutrophilic alveolitis, and occurs in the setting of a preceding acute inflammatory condition (e.g. pneumonia, non-pulmonary sepsis, acute pancreatitis, trauma, etc). Ussov et al. showed that the expanded pulmonary intravascular neutrophil pool that is associated with these conditions does not by itself lead to lung injury but may promote extravascular neutrophil migration that is associated with increased alveolar permeability and therefore with lung injury.¹⁸ Increased diffuse pulmonary accumulation of neutrophils in patients with ARDS, who did not have concomitant evidence of a primary respiratory infection, was first reported by Powe et al.²⁶ Subsequently, Warshawski et al. reported that disease activity in ARDS correlated with the degree of pulmonary localization of In-111-labelled neutrophils recorded immediately after injection, indicating increased intravascular trapping, and critically also at 17–20 h, indicating subsequent extravascular migration.²⁷ In a later study, increased diffuse FDG uptake in the lungs of patients with blunt thoracic trauma and pulmonary contusion, consistent with lung neutrophil accumulation, was found to be predictive of ARDS.²⁸ The above findings were distilled by Singh et al. into the hypothesis that the healthy pulmonary vasculature selectively traps primed neutrophils, facilitates their de-priming and later releases them into the systemic circulation in a quiescent state,²⁹ but when this host defence mechanism fails, neutrophils migrate into the lung interstitium and cause ARDS. This hypothesis was experimentally supported by the trafficking studies of ex vivo primed granulocytes undertaken by Summers et al.¹

Bronchiectasis

In health, In-111-labelled neutrophils are destroyed in the reticuloendothelial system largely by apoptosis, and the In-111 is transferred to and stably retained in macrophages.³⁰ The biological turnover of In-111 is therefore slow with a half-life of several days and a whole-body loss of <10% over 5 days. When In-111-labelled neutrophils migrate into an enclosed abscess, they undergo local apoptosis and efferocytosis, or remain intact for prolonged periods; either way, the label is retained within the location of the abscess. In inflammation involving tracts that allow drainage, however, such as in bronchiectasis and IBD, the label, either detached or still in neutrophils, travels distally within the tract and is excreted.^31,32 Neutrophils may migrate into the airways in large numbers in bronchiectasis, and in severe disease give positive images at 3–4 h following injection of In-111-labelled neutrophils. Over the course of a few days, the activity within the bronchiectatic segments clears and is excreted, with the result that sputum contains abundant radioactivity.³¹ On delayed imaging, activity can therefore be seen in the intestines arising from swallowed sputum. The excretion of In-111 provides the opportunity for quantifying disease activity by measuring the whole-body retention of In-111 using either an uncollimated gamma camera³¹ or dedicated whole-body counter. In severe disease, and similarly to severe IBD, <50% of the administered In-111 remains within the body at 5 days compared with >90% in healthy individuals.

Lobar pneumonia

Acute lobar pneumonia is negative on leucocyte scanning unless the labelled cells are injected within 24–48 h of onset, after which neutrophil migration is ‘switched off’ and resolution commences.³³ In contrast, accumulation of FDG in the consolidated lung continues beyond 48 h,³⁴ presumably because the migrated cells are metabolically active and FDG, as a small diffusible molecule, has access to them. If migration fails to switch off, then the pneumonia is at risk of progressing to a suppurative pneumonia with lung abscess, and the leucocyte scan remains positive. In bronchiectasis, the situation is reversed in that leucocyte scanning is positive but FDG PET is negative,³⁴ presumably because, once in the airways, the neutrophils have only limited access to FDG.

Chronic obstructive pulmonary disease

Chronic obstructive pulmonary disease (COPD) is seen primarily in cigarette smokers. Active components within cigarette smoke interact with cells of the innate immune system, causing the release of pro-inflammatory cytokines and chemokines. Neutrophils are the most abundant inflammatory cell present within the airways. Histotoxic molecules, such as myeloperoxidase and neutrophil elastase, released following neutrophil degranulation, play a significant role in the pathogenesis of COPD.³⁵ The presence of In-111 in the sputum of COPD patients following injection of In-111-labelled neutrophils indicates migration of neutrophils from blood to the airways. Activity levels peak at 24 h post-injection, falling to baseline by 48 h.²

In contrast to focal inflammatory lung disease, functional data on the accumulation of neutrophils in COPD has been difficult to obtain, largely due to the global nature of the disease. Moreover, physiological uptake of labelled neutrophils in bone marrow of the chest wall interferes with the lungs on planar imaging, although this can be overcome using SPECT. Accordingly, Ruparelia et al. measured accumulation of Tc-99m-labelled neutrophils in the lungs of six COPD patients from multiple sequential SPECT scans acquired over a 4-hour period.² ROI were drawn on these images, with exclusion of chest wall, and Gjedde–Patlak-Rutland analysis used to determine global clearance of labelled neutrophils from blood into lungs, expressed in units of ml/min/ml marginated neutrophil pool. There was a higher clearance rate of labelled neutrophils into the lungs of COPD patients (0.0073–0.03 ml/min/ml) compared to healthy volunteers (0–0.002 ml/min/ml), but no difference was seen between current and ex-smokers in the COPD group.²

Whole-body profiling in COPD patients and healthy controls injected with ultra-low doses of In-111-labelled neutrophils revealed initial uptake of radioactivity in marginating pools of liver and spleen, but by 24 h activity had redistributed to bone marrow for physiological disposal (Fig. 1).³⁶ There was no significant difference in whole-body In-111 loss between the two groups. Mean 7-day whole-body loss was similar in healthy non-smokers (5.5%) and all COPD patients (7.0%). Current COPD smokers, however, had a significantly higher 7-day whole-body loss (8.0%) compared with healthy non-smokers.

Mean whole-body In-111-loss in COPD was <1% per day, lower than previously seen in active IBD and bronchiectasis.² Although the reasons for this are not clear, the resting bio-distribution of neutrophils in COPD appears to be abnormal. Thus, recent data showed an increase in the marginating pool of neutrophils within the liver and, more significantly, the spleen in stable stages 2 and 3 COPD patients compared with healthy controls³⁷, suggesting that in COPD neutrophils behave non-physiologically, even in the resting state. This may be the result of neutrophil priming, which has been described in COPD.³⁸ Changes similar to those seen in liver and spleen in COPD may also take place in other systemic inflammatory diseases associated with priming, but there are no data in relation to this.

Asthma

Asthma is a heterogeneous disorder characterized by different phenotypes and endotypes.^39,40. The phenotype traditionally associated with asthma is the eosinophilic, allergic phenotype, driven by a Th2 immune response, and is responsive to corticosteroids. The ability to correctly phenotype and endotype patients, as well as localize and quantify eosinophilic inflammation, is becoming increasingly important, both clinically in the management of asthma and for testing the efficacy of novel therapeutics. Existing biomarkers of eosinophilic inflammation are indirect or invasive,⁴¹ so there is a need for less invasive methods to localize eosinophilic inflammation in the lung, as reviewed by Castro et al.⁴²

Lukawska et al.^43,44 used dynamic planar gamma camera imaging to determine lung transit times of both Tc-99m-labelled neutrophils and Tc-99m-labeled eosinophils, separated using magnetic beads. They demonstrated delayed transit of neutrophils relative to eosinophils in healthy individuals and asthmatics. Neutrophils, however, were positively selected and hence probably activated. Similarly, their purified eosinophils displayed upregulation of the activation marker, CD69.

SPECT/CT and dynamic planar imaging were used by Farahi et al. to quantify eosinophil migration into the lungs and localize focal eosinophilic inflammation following injection of Tc-99m-labelled eosinophils in nine healthy subjects (Fig. 4A and B), 10 asthmatics and three patients with focal eosinophilic lung inflammation.^45,46 Dynamic imaging over 40 min was followed by three sequential SPECT scans up to 9 h post-injection. Serial blood samples were taken and Gjedde–Patlak-Rutland graphical analysis used to quantify blood Tc-99m-eosinophil clearance into the lungs. This was multiplied by the peripheral blood eosinophil count to give eosinophil migration into the lungs in absolute numbers. Focal eosinophilic inflammation was detected in the lung nodules and foot ulcers of a patient with vasculitis,⁴⁵ and in the lungs of a patient with probable IgG4-related disease (Fig. 4C).⁴⁷ There were 4-fold and 130-fold increases in eosinophil migration in asthmatics and patients with focal inflammation, respectively, compared with healthy subjects. Thus, labelled eosinophil scintigraphy, coupled to SPECT/CT, is able to localize eosinophilic inflammation and quantify eosinophil migration into the lungs and, like labelled neutrophils in COPD, has potential for testing novel therapeutics.

Fig. 4.

Distribution of radiolabelled eosinophils in healthy controls and a patient with eosinophilic vasculitis. (A) Representative planar gamma camera images acquired after injection of Tc-99m-labelled eosinophils, isolated by negative magnetic bead selection in a healthy volunteer. The images are composites from a dynamic imaging sequence captured between 0–5, 15–20 and 35–40 min post-injection and show accumulation of tracer in the right lung (RL), left lung (LL), liver (L) and spleen (S). (B) Representative transaxial, coronal and sagittal co-registered SPECT/CT images from a healthy volunteer 45 min following injection of Tc-99m-labelled eosinophils. Images show physiological accumulation of activity in bone marrow of spine (BM), liver (L) and spleen (S). (C) SPECT/CT image 45 min following injection of Tc-99m-labelled eosinophils in a patient with eosinophilic vasculitis, showing accumulation of activity in lung nodules in the right lung (white arrows). Panel C was published by Farahi et al.⁴⁵

Conclusions and future projections

Planar and 3D-SPECT/CT imaging of radiolabelled leucocytes is now firmly embedded in clinical practice. Many nuclear medicine units are therefore well set up to use mixed leucocytes or leucocyte subsets for other cell-labelling applications. Recent advances in SPECT/CT technology are also driving better and faster image acquisition, and concomitant anatomical imaging is allowing precise information on cell localization. Methods for purifying myeloid cells and labelling have also advanced and the historic problems associated with ex vivo cell priming and activation, which impact markedly on the physiological behaviour of these cells when returned to the circulation, have also been largely overcome. In the near future, therefore, it will be possible to track and quantify myeloid cell uptake, for example in tumours to guide immunotherapy. Moreover, use of radiolabelled neutrophils to determine rates of whole organ uptake in real time should help to design smart proof-of-concept phase-two studies to determine the efficacy of novel neutrophil (and other myeloid cell)-targeted treatments. Radiolabelled eosinophils have the potential to define the extent of post-radiotherapy colitis, identify eosinophil-dominant asthma endotypes, and aid in the diagnosis of conditions such as EGPA (Churg–Strauss Syndrome). Monocyte labelling is currently being refined and will lead to intense research on the trafficking of monocytes. The remaining outstanding challenges that once tackled will transform experimental medicine and diagnostic approaches across a wide range of inflammatory and autoimmune conditions are firstly development of cell labelling with PET tracers and secondly the tracking of lymphocytes and lymphocyte subsets. Cell labelling and imaging is here to stay and is set to have a continuing role in diagnostic imaging, in patient phenotyping and assessment and molecular targeting of inflammation.

Funding

The authors gratefully acknowledge funding support from Asthma UK [08/11], the Medical Research Council [grant number MR/J00345X/1], the Wellcome Trust [grant number 098351/Z/12/Z], AstraZeneca, GlaxoSmithKline and National Institute for Health Research (Cambridge Biomedical Research Centre Cardiovascular and Respiratory Theme).

Conflict of interest statement

The authors have no potential conflicts of interest.